Indirect immunofluorescent antibody (IFA), latex agglutination (IA), and enzyme immunoassay (EIA) methods were compared for evaluation of the serum antibody responses of dogs experimentally and naturally exposed to spotted fever-group rickettsiae. Selected sera (obtained on days 1, 42, 53, 124, 145, 236, 255, 264, and 292) were examined from three 8-month-old female Beagles inoculated with Rickettsia rickettsii on days 34 and 250 of the study. A second group of dogs comprised three 8-month-old female Beagles inoculated with R montana on days 34 and 102. Subsequently, these dogs were inoculated with R rickettsii on day 250. Serum samples were obtained from the second group of dogs on days 1, 96, 103, 132, 180, 215, 292, and 494. A third group consisted of 21 naturally exposed dogs, from which sequentially obtained serum samples were available, and which had clinical signs compatible with Rocky Mountain spotted fever. Clinical signs of disease in dogs of the third group resolved after treatment with tetracycline (22 mg/kg of body weight, Po, q 8 h) was instituted. At least 2 sequentially obtained serum samples from each dog were tested. In general, the first sample was obtained just prior to treatment and the convalescent serum samples were obtained at weekly or greater intervals thereafter. For correlation and reactivity data, an IFA test for IgG/IgM (using heavy and light chains-specific conjugate) was used as the reference standard for comparison of results with those of the other tests,.

The concentration of carbonic anhydrase III isoenzyme (cA-III) in serum samples from 216 clinically normal Thoroughbreds was determined by use of an enzyme immunoassay. The concentration range of cA-III was from 16.0 to 254.5 ng/ml (mean, 56.5 +/- 11.9 ng/ml). Significant differences were not detected according to age or sex. To confirm whether serum cA-III concentration was high in horses with muscle disease, serum samples of 11 horses with exertional rhabdomyolysis were analyzed by enzyme immunoassay. Their serum cA-III concentration was about 56 times (3,136 +/- 2,610 ng/ml) that of healthy Thoroughbreds. Concentration of cA-III was higher in horses with rhabdomyolysis that had been transiently recumbent than in horses with mild disease that were reluctant to move. Blood samples obtained serially from 6 horses with exertional rhabdomyolysis were studied. Serum activities of aldolase, creatine kinase, aspartate transaminase, and lactate dehydrogenase were high. Increases and decreases in concentration of cA-III were more rapid than that for aldolase, creatine kinase, aspartate transaminase, and lactate dehydrogenase activities; thus, cA-III may be clinically applicable as a diagnostic marker for muscle disease in horses.

We studied the uptake and sequential transneuronal passage of pseudorabies virus (PRV) in rat CNS by use of a combination of immunohistochemistry and in situ hybridization. Protocols for rapid detection of PRV by immunohistochemistry and in situ hybridization in rats with PRV infection of the CNS after intranasal instillation of a wild-type strain of PRV were optimized in vitro, using porcine kidney-15 cells. Pseudorabies virus-specific hybridization signals appeared in the cytoplasm and nucleus of PRV-infected porcine kidney-15 cells by postinoculation (PI) hour 6. In tissue sections of PRV-infected rats, PRV nucleic acids were detected in areas of the rat brain in close proximity to the areas in which PRV antigens were evident. The PRV was initially found in the nucleus of trigeminal ganglion neurons at PI hour 24. At PI hour 72, PRV antigens were observed in the mid-brain, and 24 hours later, in the telencephalon. We also found evidence of specific progressive transsynaptic transmission of the virus, and, on the basis of that, we have constructed a map of the synaptic contacts and pathways in the brain. Therefore, combined use of immunohistochemistry and in situ hybridization was useful for characterizing the pathogenesis of PRV in the CNS of rats after intranasal inoculation, following a pattern that mimics PRV infection of the natural host.

We generated monoclonal antibodies (MAB) against feline immunodeficiency virus (FIV) and characterized these MAB by single competition enzyme immunoassays (EIA), immunoblot analysis, and radioimmunoprecipitation. Four MAB identified 3 distinct epitopes of the FIV p24/26 gag major core protein. One MAB recognized the p16/17 gag protein none recognized envelope proteins. We developed an FIV p26 antigen capture EIA that proved more sensitive (0.5 ng of p26/ml), less expensive, and less time-consuming than reverse transcriptase assay. The same MAB were used to develop an antibody EIA specific for FIV p26. The MAB and capture assays reported should prove useful in FIV diagnosis and research.

An enzyme immunoassay (EIA) was developed for detection of dexamethasone in equine blood. Dexamethasone 21-hemisuccinate-bovine serum albumin was used for immunization of rabbits, and prednisolone 21-hemisuccinate-horseradish peroxidase was used as enzyme conjugate. The assay had sensitivity in the low-picogram range (detection limit, 0.3 pg/well, 50% inhibition of binding at 4.5 +/- 0.7 pg/well). Apart from cortisol, which was recognized by the antiserum at concentration > 8.5 ng/ml, the dexamethasone antiserum failed to interfere with endogenous steroids, but cross-reacted with triamcinolone, flumethasone, and betamethasone. Thus, the antiserum was used to perform simultaneous screening for these synthetic glucocorticoids and to confirm their identity by combining reverse-phase high-performance liquid chromatography (RP-HPLC) and EIA. The immunoreactivity obtained by direct serum measurements was characterized by means of 2 independent RP-HPLC systems. Serum extracts were submitted to RP-HPLC systems I and II, and the fractions were tested by EIA. Immunoreactive peaks were identified by comparing their retention time with that of the standard glucocorticoids used for calibration. Coinjection of an internal standard (methylprednisolone) in RP-HPLC system II yielded reproducible relative retention times. The effectiveness of the test system was evaluated, using blood from a horse treated with commonly used veterinary preparations of dexamethasone. Administration of the free alcohol of dexamethasone and of dexamethasone 21-trioxaundecanoate, both given IV, was detected, and the identity of each was confirmed for up to 48 hours. Intramuscular administration of dexamethasone 21-isonicotinate was continued for at least 14 days after injection of a therapeutic dose. The technique provided higher sensitivity and practicability than do analytic techniques currently available for glucocorticoid testing in horses and proved reliable in confirming the identity of dexamethasone, triamcinolone, flumethasone, and betamethasone in equine blood samples.

To evaluate the relationship between pneumonic lesions and distribution of bacteria, lungs from calves inoculated with Pasteurella multocida were examined histologically by use of immunoperoxidase technique. Pneumonic lesions fundamentally consisted of bronchopneumonia with fibrinopurulent pleuritis. The lesions were confirmed to be associated with inoculated P multocida, using the immunoperoxidase technique. The P multocida antigen was detected not only in the bacterial clusters in the lesions, but also in the cytoplasm of infiltrating neutrophils and macrophages. Further, immunoelectron microscopy indicated that the inoculated bacteria generally were phagocytosed and digested by neutrophils.

An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae.